Characterizing ice particles using two-dimensional reflections of a lidar beam.

We report a phenomenon manifesting itself as brief flashes of light on the snow's surface near a lidar beam. The flashes are imaged and interpreted as specular reflection patterns from individual ice particles. Such patterns have a two-dimensional structure and are similar to those previously observed in forward scattering. Patterns are easiest to capture from particles with well-defined horizontal facets, such as near-horizontally aligned plates. The patterns and their position can be used to determine properties such as ice particle shape, size, roughness, alignment, and altitude. Data obtained at Summit in Greenland show the presence of regular hexagonal and scalene plates, columns, and rounded plates of various sizes, among others.

Microsatellite markers for Amazon pellona Pellona castelnaeana (Clupeiformes: Pristigasteridae).

The Amazon pellona is one of the few species of Pristigasteridae with recognized commercial value in the Amazon. We isolated 24 and characterized 8 microsatellite loci for this species. The number of alleles ranges from 2-8 per locus. Observed heterozygosities ranged from 0.052-0.823, while expected heterozygosities from 0.052-0.836. These 8 microsatellites are potentially valuable tools for characterizing the levels and distribution of genetic diversity, population structure, and gene flow. They may also be important parameters for the genetic conservation of this species, as well as for its sister taxon Pellona flavipinnis.

Arginine deiminase PEG20 inhibits growth of small cell lung cancers lacking expression of argininosuccinate synthetase.

BACKGROUND: Some cancers have been shown to lack expression of argininosuccinate synthetase (ASS), an enzyme required for the synthesis of arginine and a possible biomarker of sensitivity to arginine deprivation. Arginine deiminase (ADI) is a microbial enzyme capable of efficiently depleting peripheral blood arginine. METHODS: Argininosuccinate synthetase expression was assessed in human small cell lung cancer (SCLC) by immunohistochemistry (IHC), with expression also assessed in a panel of 10 human SCLC by qRT-PCR and western blot. Proliferation assays and analyses of apoptosis and autophagy assessed the effect of pegylated ADI (ADI-PEG20) in vitro. The in vivo efficacy of ADI-PEG20 was determined in mice bearing SCLC xenografts. RESULTS: Approximately 45% of SCLC tumours and 50% of cell lines assessed were negative for ASS. Argininosuccinate synthetase-deficient SCLC cells demonstrated sensitivity to ADI-PEG20, which was associated with the induction of autophagy and caspase-independent cell death. Arginine deiminase-PEG20 treatment of ASS-negative SCLC xenografts caused significant, dose-dependent inhibition of tumour growth of both small and established tumours. CONCLUSION: These results suggest a role for ADI-PEG20 in the treatment of SCLC, and a clinical trial exploring this therapeutic approach in patients with ASS-negative SCLC by IHC has now been initiated.

Clostridium perfringens alpha-toxin and NetB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens.

Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C perfringens are its four major toxins (alpha, beta, epsilon, iota) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C perfingens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C perfringens alpha-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C perfringens alpha-toxin and NetB toxin that were increased in birds experimentally coinfected with Eimeria maxima and C perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.

Effect of dietary antimicrobials on immune status in broiler chickens.

This study evaluated the effects of dietary anticoccidial drugs plus antibiotic growth promoters (AGPs) on parameters of immunity in commercial broiler chickens. Day-old chicks were raised on used litter from a farm with endemic gangrenous dermatitis to simulate natural pathogen exposure and provided with diets containing decoquinate (DECX) or monensin (COBN) as anticoccidials plus bacitracin methylene disalicylate and roxarsone as AGPs. As a negative control, the chickens were fed with a non-supplemented diet. Immune parameters examined were concanavalin A (ConA)-stimulated spleen cell proliferation, intestine intraepithelial lymphocyte (IEL) and spleen cell subpopulations, and cytokine/chemokine mRNA levels in IELs and spleen cells. ConA-induced proliferation was decreased at 14 d post-hatch in DECX-treated chickens, and increased at 25 and 43 d in COBN-treated animals, compared with untreated controls. In DECX-treated birds, increased percentages of MHC2(+) and CD4(+) IELS were detected at 14 d, but decreased percentages of these cells were seen at 43 d, compared with untreated controls, while increased TCR2(+) IELs were evident at the latter time. Dietary COBN was associated with decreased fractions of MHC2(+) and CD4(+) IELs and reduced percentages of MHC2(+), BU1(+), and TCR1(+) spleen cells compared with controls. The levels of transcripts for interleukin-4 (IL-4), IL-6, IL-17F, IL-13, CXCLi2, interferon-γ (IFN-γ), and transforming growth factorß4 were elevated in IELs, and those for IL-13, IL-17D, CXCLi2, and IFN-γ were increased in spleen cells, of DECX- and/or COBN-treated chickens compared with untreated controls. By contrast, IL-2 and IL-12 mRNAs in IELs, and IL-4, IL-12, and IL-17F transcripts in spleen cells, were decreased in DECX- and/or COBN-treated chickens compared with controls. These results suggest that DECX or COBN, in combination with bacitracin and roxarsone, modulate the development of the chicken post-hatch immune system.

Impact of fresh or used litter on the posthatch immune system of commercial broilers.

This study was carried out to investigate the effects of exposure of growing broiler chickens of commercial origin to used poultry litter on intestinal and systemic immune responses. The litter types evaluated were fresh wood shavings or used litter obtained from commercial poultry farms with or without a history of gangrenous dermatitis (GD). Immune parameters measured were serum nitric oxide (NO) levels, serum antibody titers against Eimeria or Clostridium perfringens, mitogen-induced spleen cell proliferation, and intestinal intraepithelial lymphocyte or splenic lymphocyte subpopulations. At 43 days posthatch, birds raised on used litter from a GD farm had higher serum NO levels and greater Eimeria or C. perfringens antibody levels compared with chickens raised on fresh litter or used, non-GD litter. Birds raised on non-GD and GD used litter had greater spleen cell mitogenic responses compared with chickens raised on fresh litter. Finally, spleen and intestinal lymphocyte subpopulations were increased or decreased depending on the litter type and the surface marker analyzed. Although it is likely that the presence of Eimeria oocysts and endemic viruses varies qualitatively and quantitatively between flocks and, by extension, varies between different used litter types, we believe that these data provide evidence that exposure of growing chicks to used poultry litter stimulates humoral and cell-mediated immune responses, presumably due to contact with contaminating enteric pathogens.

Using Meat Labels to Communicate the Risk of Antimicrobial-Resistant Bacterial Infections from Foods of Animal Origin:: The Case for a Balanced One Health Approach to Raising Food Animals.

Consumers are increasingly confused by the numerous meat labels confronting them in the meat case. Most meat labels do not provide actionable information and many labels only add to consumer confusion. While many consumers are willing to pay a premium for products with specific attributes, the trade-offs and unintended consequences associated with various animal raising programs are not transparent and often poorly understood. Adding to this confusion is a tendency toward the use of "absence labels" on meat products that can create a negative perception of unlabeled conventional products that may or may not include the attribute in question. Communicating with consumers about the complex issue of antimicrobial resistance (AMR) is challenging. A more balanced approach to raising food animals is a new consumer choice label program based on principles of One Health that provides transparent information to consumers with mandated antibiotic stewardship practices to reduce risk of AMR originating from food animals. This holistic program strives to provide optimal health outcomes for animals, people, and the environment and avoid the negative consequences sometimes associated with more narrowly focused programs.

Retinol is essential for growth of activated human B cells.

When EBV-transformed human B cells are removed from conventional cell cultures, washed, and seeded at a low cell density in serum-free medium, their growth potential is greatly diminished. Fresh serum restores the growth of low density B cell cultures. We have traced this restorative effect to an essential factor present in the lipid fraction of serum and have identified it as all-trans retinol. The identification is based on the close similarities of the factor isolated from serum with authentic all-trans retinol as revealed by mass spectrometry, HPLC chromatography, and the ability to stimulate the growth of lymphoblastoid cells in the bioassay. Retinol is active at concentrations equal to its concentration in serum. Retinol is also a requirement for growth in suspension cultures at cell densities of 3 x 10(5)/ml. Cells removed at any time from such exponentially growing cultures and transferred to retinol-free medium cease to grow and consequently die, whereas in the continued presence of retinol, cell growth is unabated. All-trans retinal can substitute for retinol, but retinoic acid fails to stimulate the growth of lymphoblastoid cells at physiological concentrations. Normal human B lymphocytes also require retinol as a costimulator of proliferation after activation by anti-mu antibody or Staphylococcus aureus (Cowan strain) bacteria. In serum, retinol is bound to retinol-binding protein, which in turn forms a complex with prealbumin. Accordingly, we find that B cells respond to retinol bound to its physiological serum carrier, retinol-binding protein. In conclusion, human B cells are critically dependent for optimal growth in cell culture on an external supply of retinol.

Identification of NY-ESO-1 epitopes presented by human histocompatibility antigen (HLA)-DRB4*0101-0103 and recognized by CD4(+) T lymphocytes of patients with NY-ESO-1-expressing melanoma.

NY-ESO-1 is a member of the cancer-testis family of tumor antigens that elicits strong humoral and cellular immune responses in patients with NY-ESO-1-expressing cancers. Since CD4(+) T lymphocytes play a critical role in generating antigen-specific cytotoxic T lymphocyte and antibody responses, we searched for NY-ESO-1 epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules. Autologous monocyte-derived dendritic cells of cancer patients were incubated with recombinant NY-ESO-1 protein and used in enzyme-linked immunospot (ELISPOT) assays to detect NY-ESO-1-specific CD4(+) T lymphocyte responses. To identify possible epitopes presented by distinct HLA class II alleles, overlapping 18-mer peptides derived from NY-ESO-1 were synthetized and tested for recognition by CD4(+) T lymphocytes in autologous settings. We identified three NY-ESO-1-derived peptides presented by DRB4*0101-0103 and recognized by CD4(+) T lymphocytes of two melanoma patients sharing these HLA class II alleles. Specificity of recognition was confirmed by proliferation assays. The characterization of HLA class II-restricted epitopes will be useful for the assessment of spontaneous and vaccine-induced immune responses of cancer patients against defined tumor antigens. Further, the therapeutic efficacy of active immunization using antigenic HLA class I-restricted peptides may be improved by adding HLA class II-presented epitopes.

An outbreak of gangrenous dermatitis in commercial broiler chickens.

The present report describes an outbreak of gangrenous dermatitis (GD) infection in a commercial poultry farm in Delaware involving 34-day-old broiler chickens. In addition to obvious clinical signs, some GD-affected broilers also showed severe fibrino-necrotic enteritis and large numbers of Gram-positive rods in the necrotic tissue. Histopathological findings included haemorrhage, degeneration and necrosis of parenchymatous cells, especially of skin, muscle, and intestine. Immunofluorescence staining revealed Clostridium-like bacilli in the skin and the intestine. Both Clostridium perfringens and Clostridium septicum genomic sequences were identified by polymerase chain reaction in bacterial cultures isolated from the skin, muscle, and intestine, and in the frozen tissues from the GD-affected birds. Serological analysis demonstrated that both affected and clinically healthy birds from the same house had high serum antibody titres against C. perfringens, C. septicum, Eimeria, chick anaemia virus, and infectious bursal disease virus. These results are discussed in the context of the relationship between the different Clostridium spp. and the pathogenesis of GD.

Immunopathology and cytokine responses in commercial broiler chickens with gangrenous dermatitis.

Gangrenous dermatitis (GD) is an emerging disease of increasing economic importance in poultry resulting from infection by Clostridium septicum and Clostridium perfringens type A. Lack of a reproducible disease model has been a major obstacle in understanding the immunopathology of GD. To gain better understanding of host-pathogen interactions in GD infection, we evaluated various immune parameters in two groups of birds from a recent commercial outbreak of GD, the first showing typical disease signs and pathological lesions (GD-like birds) and the second lacking clinical signs (GD-free birds). Our results revealed that GD-like birds showed: reduced T-cell and B-cell mitogen-stimulated lymphoproliferation; higher levels of serum nitric oxide and alpha-1-acid glycoprotein; greater numbers of K55(+), K1(+), CD8(+), and MHC class II(+) intradermal lymphocytes, and increased K55(+), K1(+), CD8(+), TCR1(+), TCR2(+), Bu1(+), and MHC class II(+) intestinal intraepithelial lymphocytes; and increased levels of mRNAs encoding proinflammatory cytokines and chemokines in skin compared with GD-free chickens. These results provide the first evidence of altered systemic and local (skin and intestine) immune responses in GD pathogenesis in chickens.

[Characteristic of the active substance of the Saccharomyces cerevisiae preparation having radioprotective properties]. / Характеристика активной субстанции препарата дрожжей Saccharomyces сerevisiae, обладающей радиопротекторными свойствами.

The paper describes some biological features of the radioprotective effect of double-stranded RNA preparation. It was found that yeast RNA preparation has a prolonged radioprotective effect after irradiation by a lethal dose of 9.4 Gy. 100 % of animals survive on the 70th day of observation when irradiated 1 hour or 4 days after 7 mg RNA preparation injection, 60 % animals survive when irradiated on day 8 or 12. Time parameters of repair of double-stranded breaks induced by gamma rays were estimated. It was found that the injection of the RNA preparation at the time of maximum number of double-stranded breaks, 1 hour after irradiation, reduces the efficacy of radioprotective action compared with the injection 1 hour before irradiation and 4 hours after irradiation. A comparison of the radioprotective effect of the standard radioprotector B-190 and the RNA preparation was made in one experiment. It has been established that the total RNA preparation is more efficacious than B-190. Survival on the 40th day after irradiation was 78 % for the group of mice treated with the RNA preparation and 67 % for those treated with B-190. In the course of analytical studies of the total yeast RNA preparation, it was found that the preparation is a mixture of single-stranded and double-stranded RNA. It was shown that only double-stranded RNA has radioprotective properties. Injection of 160 µg double-stranded RNA protects 100 % of the experimental animals from an absolutely lethal dose of gamma radiation, 9.4 Gy. It was established that the radioprotective effect of double-stranded RNA does not depend on sequence, but depends on its double-stranded form and the presence of "open" ends of the molecule. It is supposed that the radioprotective effect of double-stranded RNA is associated with the participation of RNA molecules in the correct repair of radiation-damaged chromatin in blood stem cells. The hematopoietic pluripotent cells that have survived migrate to the periphery, reach the spleen and actively proliferate. The newly formed cell population restores the hematopoietic and immune systems, which determines the survival of lethally irradiated animals.

From farm management to bacteriophage therapy: strategies to reduce antibiotic use in animal agriculture.

To reduce the use of antibiotics in animal agriculture, a number of effective or commercially viable alternatives have been implemented by food animal producers or are under development. Perhaps the most well-established strategies are flock and herd management practices to mitigate disease introduction and spread, and, subsequently, reduce the need for antibiotic use. While vaccines in food animal production have been used to prevent both bacterial and viral diseases, but historically, most vaccines have targeted viral diseases. Though vaccines against viral diseases can help reduce the need for antibiotic use by controlling the spread of secondary bacterial infections, more recent vaccines under development specifically target bacteria. New developments in selecting and potentially tailoring bacteriophages provide a promising avenue for controlling pathogenic bacteria without the need for traditional small-molecule antibiotics. In this article we discuss these established and emerging strategies, which are anticipated to reduce the reliance on antibiotics in food animal production and should reduce the prevalence and transmission to humans of antimicrobial resistant bacteria from these systems.

Antimicrobial-resistant bacterial infections from foods of animal origin: understanding and effectively communicating to consumers.

Consumers are increasingly interested in the attributes of the food they consume. This includes what is in the food and how it was raised; and at least some consumers are willing to pay a premium for products with specific attributes. However, the current plethora of labels on the market does not adequately address this issue; rather than providing actionable information, most labels add to the consumer confusion. In addition, there is a tendency toward "absence labels" that can contribute to a negative consumer perception of conventional products that may or may not include the attribute in question. Communication with consumers about the complex and highly technical issue of antimicrobial resistance (AMR) is challenging, and experiences from communication efforts about food safety-related issues demonstrate exactly how challenging this is to communicate clearly. General lessons learned from the science of risk communication can help guide efforts to communicate about the challenging issue of AMR. There are efforts underway to chart out a new approach. A new labeled animal production certification program is under development to provide choice for consumers, while reducing consumer confusion, which mandates antibiotic stewardship practices.

Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

Characterization of IgG and IgM antibodies induced in melanoma patients by immunization with purified GM2 ganglioside.

The ganglioside GM2 is a differentiation antigen expressed on the cell surface of human malignant melanomas and other cancers of neuroectodermal origin. We have previously reported that immunization with purified GM2 combined with Bacillus Calmette-Guérin as adjuvant and pretreatment with low-dose cyclophosphamide induced production of antibodies against GM2 in five of six patients. We have now extended our study and analyzed the induced antibodies against GM2 were not detected. After immunization, high-titer IgM antibodies were induced in 17 of 24 patients, and high-titer IgG antibodies in eight cases. Additional treatment of 12 patients with cimetidine, a histamine H2 receptor antagonist reported to have antisuppressor cell activity, had no effect on GM2 antibody titers. Antibodies against asialo-GM2 were present in all patients, and antibodies against GM1 were present in 33% of patients, before and after immunization. Antibodies induced by immunization were specific for GM2, though some reacted predominantly with N-acetylneuraminic acid GM2 (GM2), and some reacted with GM2 and N-glycolylneuraminic acid GM2(NeuGcGM2). The pattern of reactivity with GM2 is consistent with the response to T-cell-independent antigens: both IgM and IgG antibody responses against GM2 were short lived; peak titers seen after initial and secondary vaccinations were similar; and delayed-type hypersensitivity responses to skin test challenges with GM2 were not detected in any patients. However, the IgG response typed as predominantly IgG1 and IgG3, not IgG2 as might be expected for carbohydrate antigens (which are generally T-cell-independent antigens). Because IgG1 and IgG3 antibody responses are usually to T-cell-dependent antigens, the humoral immune response elicited by GM2 vaccination has both T-cell-dependent and T-cell-independent characteristics. These IgM and IgG responses against this neuroectodermal differentiation antigen expressed on melanoma cells have been induced without evidence of neurological or other toxicity.

Biochemical and serological characteristics of natural 9-O-acetyl GD3 from human melanoma and bovine buttermilk and chemically O-acetylated GD3.

Because its expression appears to be largely restricted to human melanomas, 9-O-acetyl-GD3 is a candidate antigen for vaccine construction. Searching for potential sources, we compared chemically O-acetylated calf brain GD3 and 9-O-acetyl-GD3 extracted from bovine buttermilk with 9-O-acetyl-GD3 from human melanoma. Three fractions (F1-F3) of chemically O-acetylated GD3 differed in the number and position of O-acetyl groups. O-Acetylation sites were the lactose portion in F1 and lactose as well as sialic acid in F2 and F3. Natural (melanoma- or buttermilk-derived) 9-O-acetyl-GD3 was O-acetylated solely on the sialic acid moiety. While F1 was not reactive with monoclonal antibodies against 9-O-acetyl-GD3, F2 and F3 were as reactive as the natural products. Immunization with the natural products induced high-titer antibodies against natural 9-O-acetyl-GD3 as well as F2 and F3. In contrast, mice immunized with the synthetic fractions produced antibodies only against the immunogen but not against natural 9-O-acetyl-GD3. Only immunization with the natural products induced production of antibodies reactive with surface antigens of melanoma cells expressing 9-O-acetyl-GD3. The findings suggest (a) that C-9 of the subterminal sialic acid is the site of chemical O-acetylation in F2 and F3, as opposed to C-9 of the terminal sialic acid in the natural products; (b) that O-acetylation of both the terminal and subterminal sialic acid moieties of GD3 results in recognition by three murine monoclonal antibodies (D1.1, ME 311, and Jones) reactive with human melanoma cells; (c) that O-acetylation of the terminal sialic acid is critical, on the other hand, for inducing an immune response against melanoma 9-O-acetyl-GD3; and (d) that O-acetyl GD3 from bovine buttermilk can substitute as immunogen for inducing an immune response against human melanoma cell surface antigens in the mouse.

Monoclonal antibody 806 inhibits the growth of tumor xenografts expressing either the de2-7 or amplified epidermal growth factor receptor (EGFR) but not wild-type EGFR.

The monoclonal antibody (mAb) 806 was raised against the delta2-7 epidermal growth factor receptor (de2-7 EGFR or EGFRvIII), a truncated version of the EGFR commonly expressed in glioma. Unexpectedly, mAb 806 also bound the EGFR expressed by cells exhibiting amplification of the EGFR gene but not to cells or normal tissue expressing the wild-type receptor in the absence of gene amplification. The unique specificity of mAb 806 offers an advantage over current EGFR antibodies, which all display significant binding to the liver and skin in humans. Therefore, we examined the antitumor activity of mAb 806 against human tumor xenografts grown in nude mice. The growth of U87 MG xenografts, a glioma cell line that endogenously expresses approximately 10(5) EGFRs in the absence of gene amplification, was not inhibited by mAb 806. In contrast, mAb 806 significantly inhibited the growth of U87 MG xenografts transfected with the de2-7 EGFR in a dose-dependent manner using both preventative and established tumor models. Significantly, U87 MG cells transfected with the wild-type EGFR, which increased expression to approximately 10(6) EGFRs/cell and mimics the situation of gene amplification, were also inhibited by mAb 806 when grown as xenografts in nude mice. Xenografts treated with mAb 806 all displayed large areas of necrosis that were absent in control tumors. This reduced xenograft viability was not mediated by receptor down-regulation or clonal selection because levels of antigen expression were similar in control and treated groups. The antitumor effect of mAb 806 was not restricted to U87 MG cells because the antibody inhibited the growth of new and established A431 xenografts, a cell line expressing >10(6) EGFRs/cell. This study demonstrates that mAb 806 possesses significant antitumor activity.

Ny-ESO-1 expression and immunogenicity associated with transitional cell carcinoma: correlation with tumor grade.

NY-ESO-1 mRNA expression in transitional cell carcinoma was investigated by reverse transcription-PCR and immunohistochemistry. NY-ESO-1 mRNA was detected in 20 of 62 (32%) tumor specimens. There was a correlation between NY-ESO-1 expression and tumor grade: 0 of 4 (0%) grade 1 (G1), 6 of 26 (23%) grade 2 (G2), and 14 of 32 (44%) grade 3 (G3) tumors were NY-ESO-1 mRNA positive. Immunohistochemical analysis using NY-ESO-1-specific monoclonal antibody ES121 showed that 2 of 14 NY-ESO-1 mRNA-expressing G3 tumors were positive for NY-ESO-1. No NY-ESO-1 staining was observed in the panel of 30 G1 or G2 tumor specimens, including 6 NY-ESO-1 mRNA-positive cases. Sera from an expanded panel of 124 patients with transitional cell carcinoma were tested for the presence of NY-ESO-1 antibody. Seropositivity was observed in 9 of 72 (12.5%) patients with G3 tumors, whereas none of 52 patients with G1 or G2 tumors produced antibody against NY-ESO-1. In the 9 positive patients with NY-ESO-1 antibody, 4 had muscular invasive tumors, and 5 had carcinoma in situ.

Specific localization, gamma camera imaging, and intracellular trafficking of radiolabelled chimeric anti-G(D3) ganglioside monoclonal antibody KM871 in SK-MEL-28 melanoma xenografts.

The chimeric monoclonal antibody KM871, directed against the G(D3) antigen, is under evaluation for its potential to target melanoma. To facilitate the in vivo evaluation of biodistribution properties and measurement of pharmacokinetics, KM871 was radiolabeled with (125)I via tyrosine residues and with (111)In via the bifunctional metal ion chelator C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A"-DTPA) to lysine residues. Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconjugates. Binding was enhanced in the presence of added unlabeled antibody. A humanized A33 antibody was similarly labeled with the two isotopes and used as a control. To determine and compare in vivo biodistribution characteristics of KM871 radiolabeled with (111)In or (125)I, mixtures of the radioconjugates were injected i.v. into BALB/c nude mice bearing G(D3)-positive-SK-MEL-28 melanoma xenografts. Gamma camera images were acquired; groups of five mice were sacrificed at various time intervals, and tumors, blood, and tissues were analyzed. (111)In-labeled CHX-A"-DTPA-KM871 showed a maximum tumor uptake of 41.9 +/- 7.0% injected dose/g at 72 h with prolonged retention over a 15-day period. The tumor:blood ratio was 3:1 by 72 h, and higher ratios were observed at later time points. No abnormal accumulation of (111)In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of (125)I-labeled KM871 in the same tumors. The specificity of antibody localization was confirmed by the low tumor uptake values for radiolabeled control antibody. Gamma camera imaging demonstrated excellent uptake of (111)In-labeled CHX-A"-DTPA-KM871 in the xenografts. Chromatographic analyses of xenograft cytosolic extracts demonstrated tumor internalization and catabolism of radiolabeled KM871 with the formation of small molecular weight metabolites. Laser scanning confocal microscopy demonstrated that the majority of intracellular KM871 is localized to lysosomes. Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tumor localization was shown through immunohistochemical examination of xenograft biopsies. This study demonstrates for the first time the in vivo localization of a radiolabeled anti-G(D3) monoclonal antibody to G(D3)-expressing xenografts using gamma camera scanning techniques and tumor cell internalization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, through confocal microscopy. KM871 has potential for targeting tumors in patients with melanoma.